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Enantioselective Imidation of Sulfides via Enzyme-Catalyzed Intermolecular Nitrogen-Atom Transfer Christopher C. Farwell,‡ John A. McIntosh,‡ Todd K. Hyster, Z. Jane Wang, and Frances H. Arnold* Division of Chemistry and Chemical Engineering 210-41, California Institute of Technology, 1200 East California Blvd, Pasadena, California 91125, United States S Supporting Information *
ABSTRACT: Engineering enzymes with novel reaction modes promises to expand the applications of biocatalysis in chemical synthesis and will enhance our understanding of how enzymes acquire new functions. The insertion of nitrogen-containing functional groups into unactivated C−H bonds is not catalyzed by known enzymes but was recently demonstrated using engineered variants of cytochrome P450BM3 (CYP102A1) from Bacillus megaterium. Here, we extend this novel P450-catalyzed reaction to include intermolecular insertion of nitrogen into thioethers to form sulfimides. An examination of the reactivity of different P450BM3 variants toward a range of substrates demonstrates that electronic properties of the substrates are important in this novel enzyme-catalyzed reaction. Moreover, amino acid substitutions have a large effect on the rate and stereoselectivity of sulfimidation, demonstrating that the protein plays a key role in determining reactivity and selectivity. These results provide a stepping stone for engineering more complex nitrogenatom-transfer reactions in P450 enzymes and developing a more comprehensive biocatalytic repertoire.
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INTRODUCTION irect oxidation of unactivated C−H bonds and heteroatoms in small molecules is a valuable method for introducing complexity into industrially and medically important compounds.1 Modification of sulfur heteroatoms is particularly interesting given the potential for chirality in trivalent sulfur compounds and the efficacy of chiral sulfoxide therapeutics.2 Enzymes catalyze a wide range of reactions in nature, and oxygenases in particular are powerful catalysts of heteroatom oxidation and C−H bond activation.3 Enzymes capable of catalyzing sulfoxidation are well documented4 and have even found industrial application,5 underscoring the utility of biocatalysis in heteroatom functionalization. The nitrogen analogues of sulfoxides, known as sulfimides (Figure 1), are useful building blocks in chemical synthesis,6 ligands for metal catalysts,7 and are functional groups in agrochemicals.8 Subsequent oxidation to form sulfoximines can be achieved with good chirality transfer,9 and the resulting compounds are a promising source of novel derivatives of therapeutic small molecules.10 Furthermore, sulfides substituted at the sulfur position with allyl groups can undergo a 2,3sigmatropic rearrangement upon sulfimidation, resulting in formation of a new C−N bond.11a Available methods for producing sulfimides are largely based on organometallic catalytic systems using iron or rhodium, with only one example of an iron-based asymmetric sulfimidation catalyst reported.9,11 These methodologies typically require iminoiodinane nitrene precursors and chiral ligand frameworks to achieve stereoselectivity. Transition-metal catalysts for nitrenoid transfer also
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© 2014 American Chemical Society
Figure 1. (A) P450-catalyzed sulfoxidation, shown proceeding through compound I. This reaction can also be mediated by compound 0 (hydroperoxy intermediate). (B) Serine-ligated P411-catalyzed sulfimidation (this work), believed to proceed through a nitrenoid intermediate formed from an azide with N2 as a byproduct.
require high catalyst loadings and anhydrous conditions and may also require high temperatures.12 Enzymes offer a “green” alternative to transition-metal catalysts: they are regio- and stereoselective, nontoxic, function in aqueous media and can be produced with ease in a suitable host organism. Whereas enzymatic sulfoxidation catalysts are well-known, enzymes that catalyze the analogous sulfimidation reaction have not been reported. Our laboratory has recently shown that enzymes of the iron porphyrin-containing Received: April 15, 2014 Published: May 23, 2014 8766
dx.doi.org/10.1021/ja503593n | J. Am. Chem. Soc. 2014, 136, 8766−8771
Journal of the American Chemical Society
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the nitrene source (30 total turnovers, TTN, Figure S1). Control experiments confirmed that enzyme was necessary for sulfimide formation (Table S2). Free hemin showed no activity in this transformation. As we reported previously for intramolecular C−H amination, some of the azide was reduced to sulfonamide, in this case p-toluenesulfonamide (9).13c In other non-natural P450 reactions reported to date, it was shown that amino acid substitutions could alter both the activity and stereoselectivity of the enzymes.13a,c Thus, we tested how mutation of conserved residues C400 and T268 and other active-site residues affect sulfimidation activity (Table 1). For these experiments we used the more reactive sulfide 4methoxythioanisole, for which we measured 300 TTN with P411BM3-CIS T438S (see below for more discussion of the effect of sulfide substituents on reactivity).
cytochrome P450 family can catalyze carbenoid and nitrenoid insertion reactions with high activity and selectivity.13 Mutations to conserved residues T268 and C400 in P450BM3 from Bacillus megaterium were found to dramatically enhance reactivity for non-natural chemistry. The C400S mutation, which replaces the axial cysteine with serine, was particularly activating toward C−H amination and toward olefin cyclopropanation in whole cells.13b,c The resulting shift in the ferrous-carbon monoxide (CO) Soret band from 450 to 411 nm prompted the “P411” description for P450BM3 variants having the C400S mutation (Figure 1). These “chemomimetic” enzyme systems offer several advantages over transition-metal complexes. Because they are genetically encoded, tuning of selectivity and reactivity can be achieved through directed evolution. Moreover, these novel enzyme catalysts operate under mild, aqueous conditions amenable to sustainable chemical synthesis. Previously, we and Fasan have shown that variants of P450BM3 catalyzed the intramolecular C−H amination of arylsulfonylazides with high selectivities and hundreds of total turnovers.13c,14 To further develop enzymatic nitrene transfer, we wished to explore intermolecular reactions, given the clear synthetic applications in building complexity in a convergent manner. Additionally, we reasoned that an intermolecular nitrene-transfer reaction would allow a more detailed mechanistic characterization than was possible with the intramolecular reaction. Given that variants of P450BM3 are promising enantioselective sulfoxidation and C−H amination catalysts, we sought to determine whether P450 variants could catalyze the nitrogen analogue of sulfoxidation, the intermolecular insertion of nitrogen into organosulfur compounds. This study describes the first report of intermolecular nitrenetransfer catalyzed by an enzyme, in the context of imidation of organosulfur compounds.
Table 1. Sulfimidation Activity and Selectivity of BM3 Variants Using Substrates and Reaction Conditions Showna
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RESULTS AND DISCUSSION In our previous work on intramolecular C−H amination, we were limited to aryl sulfonylazide substrates as nitrene precursors. Despite the success with this substrate class, we wished to assess the influence of the R-group on the nitrenoid transfer and thus tested a series of substrates displaying a range of stereoelectronic properties that have been shown to be effective nitrene precursors in other contexts (Figure S1, 1− 6).11b,12 For the thioether acceptor substrate we chose thioanisole, which has been used in enzymatic sulfoxidation by cytochrome P450s and other oxygenase enzymes.4b,d As a catalyst, we used the P411BM3-CIS T438S variant of cytochrome P450BM3, possessing the aforementioned C400S mutation. This enzyme, which contains 14 mutations relative to wild-type P450BM3 (Table S1), was previously shown to be a good catalyst in the activation of azides for intramolecular C−H insertion.13c Reaction conditions were similar to those reported previously for intramolecular C−H amination13c under anaerobic conditions with nicotine adenine dinucleotide phosphate (NADPH) supplied as a reductant. Considering the small size of reactive oxygen species naturally produced by P450s, we anticipated that smaller azides, such as mesyl azide (4) would be less sterically demanding than aryl or arylsulfonyl azides and thus a more suitable partner for reaction with thioanisole. We were thus surprised to find that of all the azides initially examined, sulfimidation activity was only observed with tosyl azide (1) as
entry
enzyme
TTN
er
1 2 3 4 5 6 7 8 9 10 11
P411BM3-CIS T438S P450BM3-CIS T438S P411BM3-CIS A268T T438S P411BM3-H2-5-F10 P411BM3-H2-A-10 P411BM3-H2-4-D4 P450BM3 P411BM3 P450BM3-T268A P411BM3-T268A P411BM3-CIS I263A T438S
300 7 19 140 84 32 10 11 19 17 320
74:26 nd nd 29:71 57:43 70:30 nd nd nd nd 18:82
“P411” denotes Ser-ligated (C400S) variant of cytochrome P450BM3.13b,c Variant IDs and specific amino acid substitutions in each can be found in the Table S1. TTN = total turnover number, er = enantiomeric ratio, nd = not determined.
a
Since activating mutations T268A and C400S were already present in P411BM3-CIS T438S, we tested the effects of reverting each mutation to the wild-type residue (Table 1, entries 1−3). Each revertant was much less active than the parent, supporting the benefit of having the C400S and T268A mutations for effective nitrene-transfer chemistry. Given the bulky nature of the aryl sulfonylazide nitrene sources and aryl thioethers, we next tested the C400S mutants of several P450BM3 variants that had been engineered via combinatorial alanine scanning to hydroxylate large substrates15 (Table 1, entries 4−6). While P411BM3-H2-5-F10 displayed comparably high levels of activity to P411BM3-CIS T438S (>100 TTN), the other mutants we tested from this library were less productive. We also tested the effects of introducing the activating mutations into wild-type P450BM3. Although these wild-type derivatives were highly active and stereoselective for intramolecular C−H amination,13c here we found that neither single mutant (T268A or C400S) nor the double mutant (T268A + C400S) were particularly active for intermolecular sulfimidation. The turnover data presented above demonstrate the sequence dependence of sulfimidation productivity. The effects, 8767
dx.doi.org/10.1021/ja503593n | J. Am. Chem. Soc. 2014, 136, 8766−8771
Journal of the American Chemical Society
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nisole (7a) gave the highest levels of activity (300 TTN). In contrast, the electron-deficient p-aldehyde substrate (7e) gave only trace amounts of sulfimide product. Further, some azides that initially appeared entirely inactive gave small amounts of sulfimide products when reacted with 4-methoxythioanisole, underscoring the importance of sulfide electronics in this reaction (Table S4). The identity of substrates also exerted a modest influence on the enantioselectivity of sulfimidation. In particular, P411BM3-CIS T438S gave er values for substrates 8a8d that ranged from 59:41 for 8c to 87:13 for 8d (Table S5, Figures S4−S6). While it is possible that some sulfides were poorer substrates due to the steric influence of the para substituent, the overall trend is strongly suggestive of electron induction to the aryl sulfide being a major contributor to activity. One notable aspect of these reactions is that significantly more sulfonamide (9) was produced when less reactive sulfides were used. Although the total turnover data suggest that sulfide electronics influence reactivity, this result could also be due to other factors, such as substrate-dependent enzyme inactivation. To assess the effect of sulfide substituents on reactivity more directly, we measured the initial rates of reaction of tosyl azide with the sulfides 7a−7d in Table 2. The initial rates correlated well with the total turnover data presented above, with p-OMe showing the highest rate of reaction (Figure S7). Given this correlation, we sought to obtain more mechanistic information by fitting the data to a Hammett plot that correlates the observed rates with each substituent’s Hammett parameter.18 We found a strong, linear relationship with a Hammett value of ρ = −4.0 (Figure 2),
however, could be due to changes in stability of the enzymes that lead to degradation over the course of the reaction. To address this possibility, we compared the initial rates of reaction using the most productive enzyme in terms of total turnover, P411BM3-CIS T438S, and the less productive P411BM3-H2-A-10 and P411BM3-H2-4-D4 enzymes (Figure S2). Differences in the total productivity (i.e., TTN) for each enzyme are mirrored in the initial rates of reaction (Table S3), suggesting that specific amino acids proximal to the heme influence binding and orientation of the substrates to effect catalytic rate enhancement. The key role of active site architecture in guiding reaction trajectory is further supported by the effects of amino acid substitutions on the reaction stereochemistry. Indeed, we found enzymes capable of producing an excess of either sulfimide enantiomer: e.g., P411BM3-CIS T438S gave an er of 74:26, while expanded active site variant P411BM3-H2-5-F10 exhibited the opposite selectivity, giving 29:71 (Figure S3). Among the H2 mutants (which differ from P411BM3-CIS T438S by 3−5 amino acid substitutions, Table S1), H2-5-F10 was alone in containing the I263A mutation, suggesting this mutation is responsible for enantiomeric inversion observed in the P411BM3-H2-5-F10 variant. When the I263A mutation was placed in the P411BM3CIS background, an even more pronounced inversion in selectivity was observed (er = 18:82 for the P411BM3CIS I263A T438S variant, compared to 74:26 for P411BM3CIS T438S). This enzymatic system not only induces asymmetry in sulfimide products but also provides tunability in which selectivity can be switched with just a few mutations. Table 2. Impact of Sulfide Substituents on Sulfimidation Activity with P411BM3-CIS T438S
entry
R1 in 7
R2 in 7
TTN 8
TTN 9
a b c d e
-OMe -Me -H -H -CHO
-H -H -Me -H -H
300 190 100 30
